Novel role of RNA signaling in cross-talk between autophagy and apoptosis. During viral infections, overlapping signaling pathways regulate autophagy and apoptosis, resulting in both proviral and antiviral effects. How the cross-talk between the two evolutionarily conserved processes is integrated to determine the outcome of viral infections remains unclear and represents a critical gap in our understanding of host response to viral infection and its resolution. The long-term goal of our research is to understand the role of RNA signaling pathways in cross-regulating autophagy and apoptosis in the context of viral infections. Double-stranded RNAs produced during viral infections serve as pathogen associated molecular patterns (PAMPs) to stimulate interferon (IFN) production. The cytosolic Rig- like helicases (RLHs), endosomal Toll-like receptors (TLRs), dsRNA-dependent protein kinase (PKR) and 2'- 5'oligoadenylate synthetase (OAS) serve as dsRNA sensors in cells. It is well known that Ribonuclease L (RNase L) is activated during viral infections and generates small duplex RNAs (sdRNAs) that serve as PAMPs to amplify IFN-? production. We have recently shown that activation of RNase L also induces autophagy via stress-activated c-jun N-terminal kinase (JNK) and PKR signaling pathways. Interestingly, RNase L, JNK and PKR are also components of pro-apoptotic pathways induced by dsRNA or viral infections. This intersection of autophagy and interferon-signaling pathways which can promote apoptosis of infected cells expands the physiological roles of RNA signaling pathways in antiviral responses. Our overarching hypothesis for this proposal is that RNase L participates in cross-talk between autophagy and apoptosis by generating small duplex RNAs, affecting the outcome of viral infections. In preliminary studies we show that RNase L-cleaved RNAs inhibit autophagy and promote apoptosis by cleaving a key autophagy protein, Beclin-1. The specific aims described below include proof-of-principle studies that provide an important first step by identifying and characterizing the cloned sdRNAs. We will then determine the signaling pathways involved in regulating the switch between autophagy and apoptosis and its impact on viral infections. Aim 1: We will determine the biochemical properties of sdRNAs in promoting the cross-talk between autophagy and apoptosis. Aim 2: We will define the signaling pathways engaged by cells in promoting the cross-talk induced by sdRNAs. Aim 3: We will evaluate the physiological role of sdRNAs generated by RNase L activity in cross-regulation of autophagy and apoptosis during viral infection. We expect to identify RNA regulators that can modulate autophagy or apoptosis for therapeutic application as broad range antiviral agents. The knowledge gained from these studies is significant because they provide new insight into role of RNA signaling in pathogen sensing, autophagy and apoptosis.